Method, reagent and kit for evaluating susceptibility to premature atherosclerosis

ABSTRACT

A single point mutation in the human lipoprotein lipase gene which results in an A→G nucleotide change at codon 291 (nucleotide 1127) of the lipoprotein lipase gene, and a substitution of serine for the normal asparagine in the lipoprotein lipase gene product is seen with increased frequency in patients with coronary artery disease, and is associated with an increased susceptibility to coronary artery disease, including in particular premature atherosclerosis. This is expressed as a diminished catalytic activity of lipoprotein lipase, lower HDL-cholesterol levels and higher triglyceride levels. Thus, susceptibility of a human individual to premature atherosclerosis and other forms of coronary artery disease can be evaluated by 
     evaluating the sample of DNA for the presence of nucleotides encoding a serine residue as amino acid 291 of the lipoprotein lipase gene product. The presence of a serine residue is indicative of increased susceptibility in the patient. This method may be performed using a kit which contains a pair of primers selected to amplify a region of a human lipoprotein lipase gene spanning amino acid 291 of human lipoprotein lipase.

BACKGROUND OF THE INVENTION

This application relates to a method, reagent and kit for evaluatingsusceptibility to and causation of premature atherosclerosis and otherforms of coronary artery disease. The invention further relates to amethod of gene therapy by which lipoprotein lipase deficiencies can betreated, and to transducing vectors for use in such a method.

"Coronary artery disease" is a collective term for a variety ofsymptomatic conditions including angina, myocardial infarction, andnon-specific chest, arm and face pain, which result from atherosclerosisof the arteries that supply blood to the heart. Atherosclerosis,commonly known as "hardening of the arteries" is caused by the formationof deposits of fatty substances such as cholesterol within the innerlayers or endothelium of the arteries.

"Premature atherosclerosis" as used herein refers to the clinicalpresentation of signs and symptoms of coronary artery disease before theage of 65.

Because of the significant relationship between coronary artery diseaseand heart attacks, considerable effort has been devoted to identifyingthe biochemical causes of atherosclerosis. This research has shown thathigh levels of total cholesterol, low density lipoprotein (LDL), verylow density lipoprotein (VLDL) and triglycerides are associated withincreased risk of coronary artery disease, while high levels of highdensity lipoproteins (HDL) are associated with decreased risk ofcoronary artery disease. See, Gordon et al., The Amer. J. Med. 62:707-714 (1977). However, while observation of lipoproteins, cholesteroland triglycerides can provide a basis for identifying individuals atrisk of coronary artery disease, the levels of these substances arethemselves symptoms of an underlying biochemical defect which remainsunidentified. Thus, specific treatment of the ultimate cause rather thanan intermeditate condition, and prediction of risk prior to the onset ofthis intermediate condition is not possible through such observation.

Studies directed towards the underlying cause of coronary artery diseasehave identified a number of mutations in genes coding for proteinsinvolved in lipid transport and metabolism that appear to be associatedwith an increased risk. Examples include a large number of mutations inthe low-density lipoprotein receptor gene, Hobbs et al., Human Mutations1: 445-466 (1992), and a single mutation in the apolipoprotein-B (Apo-B)gene which underlies familial defective Apo-B in many parts of theworld. Soria et al., Proc. Nat'l Acad. Sci. USA 86: 587-91 (1989). Inaddition, mutations in other genes which play a significant role in HDLmetabolism such as the cholesterol ester transferase protein (CETP)gene, Brown et al., Nature 342: 448-451 (1989) and the gene for Apo-A1,Rubin et al., Nature 353: 265-266 (1991), have also been shown to beassociated with either enhanced resistance or increased susceptibilityto atherosclerosis. However, these mutations are uncommon and thus farno specific mutation in any gene has been found in a significant number(i.e., >1%) of patients with coronary artery disease or prematureatherosclerosis. Accordingly, these test results while interesting donot offer the opportunity to provide evaluation or therapy tosignificant numbers of patients.

SUMMARY OF THE INVENTION

It has now been found that a single point mutation in the humanlipoprotein lipase gene which results in an A→G nucleotide change atcodon 291 (nucleotide 1127) of the lipoprotein lipase gene, and asubstitution of serine for the normal asparagine in the lipoproteinlipase gene product is seen with increased frequency in patients withcoronary artery disease, and is associated with an increasedsusceptibility to coronary artery disease, including in particularpremature atherosclerosis. This is expressed as a diminished catalyticactivity of lipoprotein lipase, lower HDL-cholesterol levels and highertriglyceride levels. Thus, in accordance with the present inventionthere is provided a method for evaluating susceptibility of a humanindividual to premature atherosclerosis and other forms of coronaryartery disease comprising the steps of:

(a) obtaining a sample of DNA from the individual; and

(b) evaluating the sample of DNA for the presence of nucleotidesencoding a serine residue as amino acid 291 of the lipoprotein lipasegene product. The presence of a serine residue is indicative ofincreased susceptibility in the patient.

The invention further provides a kit for performing the method of theinvention. Such a kit comprises a pair of primers selected to amplify aregion of a human lipoprotein lipase gene spanning amino acid 291 ofhuman lipoprotein lipase. Appropriate additional reagents may also beincluded in the kit such as polymerase enzymes, nucleoside stocksolutions and the like.

A further aspect of the present invention is a method of treatingpatients suffering from or likely to suffer from prematureatherosclerosis and other forms of coronary artery disease as a resultof a lipoprotein lipase deficiency using gene therapy. This may beaccomplished using adenovirus-mediated or retrovirus-mediated genetherapy, and can be performed using either an in vivo or an ex vivoapproach.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the use of strand displacement amplification in amethod in accordance with the present invention;

FIG. 2 shows the sandwich formed when two oligonucleotide probes areused to analyze for the presence of an Asn291Ser mutation;

FIG. 3 illustrates the use of mismatch primers in accordance with theinvention to detect the Asn291Ser mutation; and

FIG. 4 shows a plasmid construct useful in accordance with the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the detection of a mutation in the genecoding for the enzyme lipoprotein lipase in a sample of DNA obtainedfrom a patient.

The first step in the method in accordance with the invention isobtaining an appropriate sample of DNA. A suitable source of such asample is from patient blood. Isolation of the DNA from the blood can beperformed by many different methods. For example, the DNA may beisolated from the leukocytes using a salt-chloroformextraction asdescribed in Trends in Genetics 5: 391 (1989).

Once the sample of patient DNA is obtained, it may be desirable toamplify a portion of the DNA including the region of interest. Onetechnique which can be used for amplification is Polymerase ChainReaction (PCR) amplification. This technique, which is described in U.S.Pat. Nos. 4,683,202 and 4,683,195, which are incorporated herein byreference, makes use of two amplification primers each of whichhybridizes to a different one of the two strands of the DNA duplex atregions which do not overlap the site of the mutation being tested for,in this case the mutation in amino acid 291. Multiple cycles of primerextension, and denaturation are used to produce additional copies of DNAto which the primers can hybridize. This amplification can be performedin a solution, or on a solid support (see, e.g. U.S. Pat. No. 5,200,314which is incorporated herein by reference).

The mutation site of interest is at a defined location within exon 6 ofthe lipoprotein lipase gene, the sequence of which is known in the art.Oka et al., Biochim. Biophys. Acta 1049: 21-26 (1990); Deeb et al.,Biochemistry 28: 4131-4135 (1989); Wion et al., Science 235: 1638-1641(1987). Amplification primers may be used which bind to the intronregions on either side of exon 6, or which bind to portions of exon 6itself. Where amplification of the mutation site is desired, the primersshould not overlap the site of the mutation of interest. Suitableprimers include those described for exon 6 in Monsalve et al., J. Clin.Invest. 86: 728-734 (1990).

Another amplification technique which may be used in accordance with thepresent invention is known as Strand Displacement Amplification (SDA).In this technique, which is described in U.S. Pat. No. 5,270,184,incorporated herein by reference, and EP 0 497 272, and which isexemplified in FIG. 1, a gene fragment is used as the target, and aprimer is used which binds to the 3'-end of this fragment. The primer isselected to include a restriction site near its 5'-end. This can beachieved by using a primer which extends beyond the 3'-end of the targetgene fragment if there is no restriction site conveniently locatedtowards the 3'-end of the fragment from the site of interest. The primerand the target fragment (if the primer extends beyond the end of thefragment) are extended to form a duplex using modified nucleosidefeedstocks, e.g., α-thio nucleoside triphosphates, at least in theregion of the restriction cleavage site so that the newly formed strandis not susceptible to cleavage by the endonuclease. For subsequentamplification normal feedstocks are used. A restriction endonuclease isintroduced which nicks the duplex at the restriction site. Extensionthen starts over at the site of the nick, at the same time that thepreviously hybridized oligonucleotide is displaced. In this way,multiple copies of one or both strands of a gene or gene fragment can beamplified without the use of temperature cycling.

To use strand displacement amplification to amplify the mutation siteresponsible for the Asn291Ser mutation, primers flanking exon 6, such asthose described in Mortsalve et al. could be used.

Once amplified, the DNA may be evaluated by any of a number of methodsto determine if the Asn291Ser mutation is present. First, the amplifiedDNA can be sequenced (optionally after cloning into a TA cloning vector,available from Invitrogen, Inc.) using manual or automated sequencing ofthe amplified product. Since the complete sequence of exon 6 of normallipoprotein lipase is known, targeted sequencing primers can be readilydeveloped for this purpose.

Another approach to the detection of Asn291Ser mutations, generally usedfollowing amplification, is the use of sequence specific oligonucleotideprobes which bind to one of the mutant or wildtype form, but not to theother. Such probes generally have a length of 15 to 20 bases. Becausethe difference being evaluated is a single base, the analysis isconducted under very stringent hybridization conditions such that onlyperfect matches will form stable hybrids.

The probe used in the invention is advantageously labeled to permit itseasy detection. Suitable labels include radioactive labels, fluorescentlabels, and reactive labels such as biotin. The probe may also belabeled twice, for example with a radiolabel and a reactive label, inwhich case the reactive label may be used to the capture the DNA hybrid,for example through the reaction of biotin with an avidin-coatedsupport.

A preferred format for testing using sequence specific probes involvesthe use of a sandwich assay in which the amplified DNA is evaluatedusing two probes. The first oligonucleotide probe is either selected tobind specifically to a gene encoding a mutant human lipoprotein lipasehaving a serine residue as amino acid 291, wherein said probe binds to aportion of the gene including the bases coding for the serine residue orselected to bind specifically to a gene encoding a normal humanlipoprotein lipase having an asparagine residue as amino acid 291,wherein said probe binds to a portion of the gene including the basescoding for the asparagine residue. The second oligonucleotide probe isselected to bind to a different, non-overlapping portion of thehuman-LPL gene which is the same in both mutant and non-mutant forms.One of the two probes is labeled with a detectable label while the otheris labeled with a reactive label to facilitate immobilization. Only whenboth probes are bound to a single piece of amplified DNA will thedetectable label be immobilized through the formation of a sandwich ofthe structure shown in FIG. 2.

Various modifications of the amplification process may also be used inaccordance with the present invention to detect the presence of anAsn291Ser mutation. If intentionally mismatched primers are used duringthe amplification, the amplified nucleic acids may also be evaluated forthe presence of the Asn291Ser mutation using a technique calledrestriction fragment length polymorphism (RFLP). In order to make use ofRFLP directly to detect a point mutation (as opposed to an insertion ordeletion mutation), the mutation must result in the addition or loss ofa site cleaved by a restriction endonuclease. If this is the case, thefragments produced upon restriction endonuclease digestion of the normaland mutant gene differ in number, in size, or in both. This differencecan be detected by gel electrophoresis of the restriction fragments.

In the case of the Asn291Ser mutation, the nucleotide sequence of thecoding strand changes from

    ______________________________________    5'- - - - - -GAC ATC AAT AAA GTC - - - - - - 3' (SEQ ID NO.: 3)    to    5'- - - - - -GAG ATC AGT AAA GTC - - - - - - 3' (SEQ ID NO.:    ______________________________________    4)

These fragments lack the two-fold symmetry that is associated withcleavage sites of restriction endonucleases, and thus one cannot simplyuse an enzyme which will cleave one of the sequences but not the other.RFLP can be used, however, if a special mismatch primer is used duringthe amplification process. This primer, shown below in Example 1, bindsto the LPL gene at a site adjacent to the mutation of interest, andintroduces an intentional error into the amplified DNA. Thus, asillustrated in FIG. 3, instead of the expected sequence, the mismatchprimer produces the duplex region

    ______________________________________    5'- - -ATAC- - -3' coding strand    3'- - -TATG- - -5' non-coding strand    when a wild-type gene is amplified, and the sequence    5'- - -GTAC- - -3' coding strand    3'- - -CATG- - -5' non-coding strand    ______________________________________

when a mutant gene is amplified, where the C/G pair in the fourthposition of the above fragments is the intentional mismatch. Amplifiedmutant genes therefore contain a restriction site (5'-GTAC-3') which iscleaved by the restriction endonuclease RsaI, but amplified wild-typesequence (5'-ATAC-3') does not. Thus, a polymorphism measurable throughrestriction fragment lengths is artificially introduced into theamplified DNA using the mismatch primers.

The amplification process may also be modified by using labeled primerswhich facilitate detection and/or capture of the amplified product. Forexample, as described in British Patent No. 2 202 328, using abiotin-labeled primer as one of the two primers permits the recovery ofthe extended primers produced during the amplification reaction, e.g.,by binding the extended primers to a support coated with (strept)avidin.If the primer used is in a region flanking the mutation site, thepresence of the mutation can be detected by adding a labeled probe,which specifically binds to the mutant or wild-type gene, to thebiotinylated amplified DNA either before or after capture of theamplified DNA on a support. If the label becomes bound to the support,this indicates that the probe was bound. Alternatively, the primer maybe one which spans the mutation site in which case amplification willoccur using a primer corresponding to the mutant sequence only when themutation is present (and vice versa). In this case, a labeled probewhich binds to a portion of the LPL gene away from the mutation site orlabeled nucleoside feedstocks may be used to introduce a label into theamplified DNA.

The presence of the Asn291Ser mutation may also be detected using acatalytic hybridization amplification system of the type described inInternational Patent Publication No. W089/09284, which is incorporatedherein by reference. Basically, in this technique, the target nucleicacid acts as a cofactor for enzymatic cleavage of probeoligonucleotides. Thus, a substantial excess of labeled probeoligonucleotide (which binds specifically to either the mutant or thewild-type gene) is combined with the target nucleic acid under stringenthybridization conditions such that only exactly complementary strandswill hybridize to any measurable extent. An enzyme is added which willcleave the probe when it is part of a duplex, but not in single strandedform. The mixture is then cycled through multiple cycles ofannealing/enzyme digestion and denaturation. If the probe binds to thetarget, the result is the production of many small labeledprobe-fragments, and the concurrent reduction in the number of full-sizelabeled probes. Either the increase in the number of fragments or thedecrease in the number of full-sized probes can be detected and providesan amplified indication of the presence or absence of the targetsequence in the sample.

An example of an enzyme which can be used in the catalytic hybridizationamplification system is RNaseH which is used in combination with RNAprobes; which are selectively cleaved when hybridized to a strand oftarget DNA. Restriction endonucleases which do not cleavephosphorothioate-modified DNA may also be used, provided that the targetDNA is first copied to produce a phosphorothioate-modified target.Because this method combines both amplification and detection, prioramplification of the genomic DNA from the sample is generally notnecessary.

Another technique useful in the present invention which combinesamplification and detection relies on the autocatalytic replication ofcertain RNA's as described in U.S. Pat. No. 4,957,858, which isincorporated herein by reference. Briefly, in this technique areplicative RNA segment is ligated to a sequence specificoligonucleotide probe which binds to either the mutant or the wild-typeform of the Asn291Ser mutation site in exon 6 of the LPL gene. Thisligated probe is then combined with the genomic DNA in such a mannerthat the probe will bind if the matching sequence is present in thegenomic DNA, and so that unbound probe can be separated from boundprobe. For example, the genomic DNA may be immobilized on a solidsupport to facilitate washing out of unbound probe molecules.Thereafter, the RNA portion of the ligated probe is amplified, forexample using the enzyme Q-beta replicase.

Yet another form of combination amplification/detection technique whichis useful in the present invention is described in U.S. Pat. No.5,124,246 which is incorporated herein by reference. In this technique,a total of five types of oligonucleotide probes are used. The first typeof probe is a multimer oligonucleotide having a "star" typeconfiguration with many generally identical arms. The second type ofprobe is a labeling probe. The labeling probe is complementary to thesequence of one of the arms of the multimer probe and includes adetectable label. The third type of probe is an immobilized probe. Aplurality of this third type of probe is affixed to a solid support. Thespecific sequences used in these first three types of probes areindependent of the nature of DNA being analyzed, except that they shouldnot hybridize with this DNA directly.

The fourth type of probe is referred to as an amplifier probe. Theseprobes are synthesized in two parts, one which is complementary to aportion of the normal sequence of exon 6 of the LPL gene away from theAsn291Ser mutation site, and one which is complementary to an arm of themultimer probe. A plurality of different types of amplifier probes isformed. These various types of probes are complementary to different,non-overlapping portions of the sequence. The fifth type of probe is acapture probe. The capture probe is also formed in two parts: one whichis complementary to the site of the Asn291Ser mutation and one which iscomplementary to the immobilized probe.

The assay is performed by combining denatured genomic DNA with theplurality of amplifier probes and capture probes under conditionspermitting hybridization. The result is the binding of numerousamplifier probes to exon 6 of the LPL gene. The capture probe will onlybind, however, if the corresponding mutant (or non-mutant, depending onthe sequence of the probe) is present. Thereafter, the solid supporthaving the third probe immobilized thereon is introduced. A solidsupport-immobilized probe-capture probe-genomic DNA-amplifier probesandwich will form if DNA complementary to the capture probe is present.The support is then washed to remove unbound material, and the multimerprobe is added. The multimer probe binds to the support via theamplification probe only if the sandwich was formed in the first place.The support is then washed and a labeling probe is added. The labelingprobe will bind to all of the available arms of the multimer probe onthe solid support, thus providing numerous detectable labels for eachactual mutation site in the DNA sample.

In the foregoing discussion of amplification and detection techniques,there is frequent mention of labeled probes or labeled primers. Forpurposes of this application, the label applied to the primer may takeany form, including but not limited to radiolabels; fluorescent orfluorogenic labels; colored or chromogenic labels; chemically reactivelabels such as biotin; enzyme-labels, for example phosphatase,galactosidase or glucosidase enzymes which can produce colored orfluorescent reaction product in combination with substrates such asp-nitrophenyl phosphate (colored reaction product) or 4-methylumbelliferyl phosphate (fluorescent cleavage product); andchemiluminescent labels.

A further aspect of the present invention is the particularoligonucleotide probes which may be used in one Or several of thetechniques as discussed above for detection of the Asn291Ser mutation.Thus, for use in the case of mismatch primer amplification followed byRFLP analysis there is provided an oligonucleotide primer which bindsspecifically to a gene encoding for human lipoprotein lipase in a regionadjacent to, but not overlapping the second base in the codoncorresponding to residue 291 in human lipoprotein lipase, and whichincludes a mismatched base which does not correspond to the normalsequence of human lipoprotein lipase, whereby upon extension of theprimer, using a target human lipoprotein lipase gene as a template, anextension product is produced which contains a restriction site whichcan be cleaved by a restriction endonuclease when the lipoprotein lipaseproduct made by the target gene has a serine residue as amino acid 291,and does not contain such a restriction site when the lipoprotein lipaseproduct made by the target gene has an asparagine residue as amino acid291. A preferred primer which binds to the coding strand is one in whicha base complementary to base number 1130 is changed from the normalthymine to guanine. For the non-coding strand, the change is fromadenine to cytosine. A particularly preferred mismatch primer forbinding to the coding strand has the sequence

    CTGCTTCTTT TGGCTCTGAC TGTA                                 (SEQ 2).

For several of the detection methods discussed above, an oligonucleotideprobe is utilized which binds to a site which includes the site of thespecific mutation of interest. Thus, the present invention encompassestwo types of oligonucleotide probes: (1) an oligonucleotide probeselected to bind specifically to a gene encoding a mutant humanlipoprotein lipase having a serine residue as amino acid 291, whereinsaid probe binds to a portion of the gene including the bases coding forthe serine residue; and (2) an oligonucleotide probe selected to bindspecifically to a gene encoding a normal human lipoprotein lipase havinga asparagine residue as amino acid 291, wherein said probe binds to aportion of the gene including the bases coding for the asparagineresidue. These probes are preferably from 15 to 20 bases in length, andmay be selected to bind to either the coding or the non-coding strand ofthe genomic DNA. Further, the probes will advantageously include adetectable label.

A further aspect of the present invention is a kit which may be used todetect the presence of the Asn291Ser mutation. The specific componentsof the kit will depend on the nature of the evaluation being conducted.In general, however, the kit will include a pair of primers selected toamplify a region of a human lipoprotein lipase gene encoding for aminoacid 291 of human lipoprotein lipase. These primers may be primers forPCR, primers adapted for strand displacement amplification, or a normalprimer and a mismatch primer. In addition, the kit may includeoligonucleotide probes for use in the detection of the Asn291Sermutation.

The discovery of the significance of the Asn291Ser mutation opens thedoor to the possibility of providing gene therapy to individuals havingthe mutation and thus to prevent or delay the onset of coronary arterydisease and particularly premature atherosclerosis. In addition, sincegene therapy to correct this defect would provide a patient with a fullyfunctional lipoprotein lipase enzyme, therapeutic agents and methodsused for this purpose may also be used effectively for other conditionsassociated with LPL mutations. Such conditions include infantile failureto thrive, hepatosplenomegaly, eruptive xanthomas, chronic and/orepisodic abdominal pain, pancreatitis and lactescent plasma due to anaccumulation of chylomicrons and very low density lipoproteins or theirremnants in the plasma.

Gene therapy to introduce functional LPL may reduce the clinicalmanifestations stemming from hypertriglyceridemia in both LPL deficienthomozygotes and heterozygotes. This gene transfer can be accomplishedusing adenovirus-DNA-polylysine conjugates; adenovirus constructs inwhich the normal LPL gene is inserted into the viral genome; orretroviral constructs in which the normal LPL gene is inserted into theviral genome. The vector may be introduced directly, for example byparenteral injection into the patient, or may be introduced via animplanted pseudo-organ.

FIG. 4 shows a plasmid construct useful in accordance with the presentinvention. As shown, the plasmid pRc/CMV-hLPL is 7.90 Kbases in size.The preparation of this particular plasmid is described below in Example2. It will be appreciated by persons skilled in the art, however, thatvariations in this technique, or the precise structure of the plasmidmay be made without departing from the present invention provided thatthe plasmid contains a functional h-LPL gene and an appropriatepromoter. For example, tissue-specific promoters, particularly adiposetissue specific or muscle specific promoters might be used in place ofthe CMV promoter. Furthermore, while the SV40 promoter and theantibiotic resistance markers are convenient for research purposes, theyare not necessary for therapeutic purposes.

To prepare a plasmid for transfection into mammalian, and particularlyhuman cells, the plasmid is complexed with an adenovirus-polylysineconjugate. In general this process involves the harvesting andpurification of a suitable adenovirus, for example a virus which isincompetent as a result of an E1A or an E3 deletion mutation. Thepurified virus is then conjugated with a polycationic material forassociating with DNA such as polylysine, polyarginine or protamine, forexample using a bifunctional reagent such as ethyl-3,3-dimethylaminopropyl carbodimide (EDC) as a crosslinking agent. When theresulting adenovirus-polylysine conjugate is combined with a plasmidcontaining a normal LPL gene, an adenovirus-DNA-polylysine complex formsspontaneously. This complex transfects mammalian cells of various typeswhen placed in media with the cells with relatively high efficiency, andthe transfected cells produce functional LPL.

Mammalian cells may also be transduced (or transfected) using anadenovirus into which a gene encoding for normal LPL has been inserted.Preferred adenoviruses are those with an E1 or an E3 deletion mutationrendering the virus incompetent. The h-LPL gene can be convenientlyinserted into the virus at the site of the deletion.

Specific modified adenoviruses useful in the present technique are basedon the RSV β-Gal adenovector described by Strafford-Perricaudet et al.,J. Clin. Invest., 90: 626-630 (1992). This adenovector is based onadenovirus Ad5. Human LPL cDNA is introduced into the vector byhomologous recombination using a modified form ofStrafford-Perricaudet's pLTRβGalpIX plasmid. The plasmid contains an RSVLTR promoter or a CMV plus intron promoter, human LPL cDNA, a poly Asite plus small intron from SV40 derived from a pSV2 vector. Mulligan etal., Science 209: 1422-1427 (1980) which are inserted betweennucleotides 455 to 3329 of an Ad5 DNA which is also deleted in the E3region. This results in the deletion of E1A and part of E1B, but, leavespIX intact. The resulting adenoviruses are non-replicating but can bepropagated in 293 cells which transcomplements the E1A activity.

A third type of vector which may be used to transduce (or transfect)mammalian cells is a retroviral vector. Suitable vectors includemyeloproliferative sarcoma virus (MPSV)-based retroviral vectors intowhich human LPL cDNA is inserted under the transcriptional control ofthe constitutive enhancer-promoter regulatory elements of the MPSV longterminal repeat (LTR).

Gene transfer vectors can be introduced into a human subject either invivo or ex vivo. In the case of an in vivo treatment, the gene transfervector may be simply injected into the patient, for exampleparenterally, and allowed to find suitable target cells. In the case ofex vivo treatment, cells are grown in vitro and transduced ortransfected with the virus, embedded in a carrier such as a collagenmatrix, which is then implanted in the patient, for example as asub-cutaneous implant. Preferred cells for use in ex vivo applicationsare fibroblast cells taken from the patient who will receive theimplant.

EXAMPLE 1

The significance of the mutation resulting in a serine in place of anasparagine as amino acid 291 in human lipoprotein lipase (the "Asn291Sermutation") was discovered as a result of a case controlled study of alarge homogeneous sample of patients undergoing diagnostic coronaryangiography. A total of 807 men, all of whom were of Dutch descent andhad angiographically proven atherosclerosis with more than 50% stenosisof at least one major coronary vessel were included in the study. All ofthe patients were less than 70 years of age, and had total cholesterollevels between 4 and 8 mmol/l and triglyceride levels which did notexceed 4 mmol/l.

The control group for the study included 157 persons who did not haveany history of angina or premature atherosclerosis, and who exhibited nosigns of vascular disease upon physical examination. The controls wereall less than 60 years of age and had baseline HDL levels greater than0.95 mmol/l and triglyceride levels of less than 2.3 mmol/l.

DNA was extracted from leukocytes using a salt-chloroform extractionmethod as described in Trends in Genetics 5: 391 (1989). Exon 6 of theLPL gene was amplified with a 5'-PCR primer located in intron 5 near the5' boundary of exon 6 having the sequence

    GCCGAGATAC AATCTTGGTG                                      (SEQ 1)

and a 3' mismatch primer which was located in exon 6 near the Asn291Sermutation. The mismatch primer had the sequence

    CTGCTTCTTT TGGCTCTGAC TGTA                                 (SEQ 2).

PCR amplification reactions were performed using 0.5 μg of genomic DNAin BRL PCR buffer containing 1.5 mM MgCl₂, 200 μM dNTPS, 1 μM eachprimer and 2.5 units Taq polymerase (BRL). The reaction mixture wasdenatured at 95° C. for 1 minute, annealed at 51° C. for 1 minute andextended at 72° C. for 45 seconds for a total of 35 cycles. Twenty μl ofthe PCR product was then digested with 10 units RsaI enzyme, 3,5 μl of10× reaction buffer 1 (BRL), and 9.5 μl of water at 37° C. for 2 hours.The digested fragments were then separated on 2% agarose gel.

Because the combination of the mismatch primer and the Asn291Sermutation produces an RsaI restriction site which is absent when themismatch primer is used to amplify the wild-type gene, the restrictionfragments observed on the agarose gel were different when the mutationwas present. Using this difference as a diagnostic indicator, it wasdetermined that the Asn291Ser mutation was seen in 41 of the 807 or5.09% of the patients in the test group, but in only 3 out of 157 or1.9% of the patients in the control group. When a subgroup of the 494patients in the test group with hypoalphalipoproteinemia was considered,it was found that a higher percentage of these patients, i.e., 6.9% (34out of 494) had the Asn291Ser mutation. When a further subgroup of thetest group was considered by selecting those individuals with low HDL-Clevels (<1.0%), and excluding those individuals who had bloodglucose >6.8 mmol/l (suggestive of diabetes) and those on β-blockertherapy, 11.3% (12 out of 106 patients) had the mutation. Thisproportion further increased when those with still lower HDL-C levelswere considered separately. Thus, among persons with HDL-C levels lessthan 0.9 mmol/l, 8 out of 68 or 12.5% had the Asn291Ser mutation, whileamong those with HDL-C levels less than 0.8 mmol/l, 5 out of 32 or 15.6%had the Asn291Ser mutation.

EXAMPLE 2

pRc/CMV vector (Invitrogen) was linearized using XbaI and Hind III. AnXbaI/HindIII fragment containing h-LPL cDNA having a length of about 2.4kb was inserted into the vector. DH5-alpha was transformed with theconstruct. Transformed cells were selected from agar plates based uponampicillin resistance, and grown in LB medium. The plasmid construct,pRc/CMV-hLPL which is shown in FIG. 4, was isolated from the cultures byalkaline lysis and CsCl centrifugation.

EXAMPLE 3

A purified preparation of an incompetent adenovirus (E1A deletionmutant) was prepared by growing 293 cells in 2 liter spinner flasks to acell density of 4.5×10⁶ /ml and infecting the cells with DL312adenovirus stock at MOI (multiplicity of infection) 20-50 for 1 hour.Forty hours post infection, the cells were harvested by centrifugation.A lysate was prepared by subjecting the harvested cells to 3 freeze/thawcycles. This lysate was centrifuged in a two-layer CsCl gradient(d=1.25, d=1.4) in a Beckman SW41 swing rotor at 35,000 rpm and 18° C.for 90 minutes. After the ultracentrifugation, the virus was recoveredfrom the interface between the two CsCl layers using a syringe and along needle. The recovered virus was then placed onto a CsCl solution(d=1.34) and centrifuged for 16 hours at 35,000 rpm and 18° C. Afterthis centrifugation, the virus was again recovered from the interfaceand was then dialyzed three times (1 hour per cycle) against a sterilebuffer (Tris 10 mM, MgCl₂ 1 mM, NaCl 0.135M). In the third dialysiscycle, the buffer included 10% glycerol to enhance storage stability.The purified virus was kept frozen at -80° C. until ready to use.

EXAMPLE 4

Virus prepared as described in Example 3 was mixed with polylysine (10mM) and EDC (2 mM) for 4 hours at 4° C. in HBS/buffered saline to formadenovirus-polylysine conjugates. The conjugates were re-isolated byCsCl gradient centrifugation using the same protocol as the finalcentrifugation in Example 3.

The re-isolated conjugates (5×10⁹ /ml) were incubated with 60-70%confluent Chinese Hamster Ovary cells (CHO K-1) in 2% FBS medium (1 ml)and 6 μg of the plasmid pRc/CMV-hLPL. As a control to assess the extentto which transfection occurred, a second set of samples was prepared inthe same manner using the plasmid pRc/CMV-B-gal which includes a geneencoding β-galactosidase in place of h-LPL. After two hours, the mediumcontaining the conjugates was aspirated out, and new medium (10% FBS)was added to the cells.

By incubating the control cells infected with pRc/CMV-B-gal in thepresence of X-gal, and counting the number of cells which evidenced thecharacteristic blue color which result from cleavage of X-gal byβ-galactosidase, it was determined that the transfection efficiency inthis system varied from 2% when the virus solution was diluted 2000× to50% when the virus solution was diluted 125×. Thus, 50 % transfectionefficiency could be achieved in vitro at titers of 0.5-1×10⁸, which isat least 10-fold less than the titers which would normally be used invivo.

To determine the expression of LPL in cells transfected withpRc/CMV-LPL, the activity of LPL was determined and compared to theactivity observed for control cells transfected with pRc/CMV-B-gal. Forthe control cells, the activity measured was 12 mU/ml. For the cellstransfected with pRc/CMV-LPL, the activity measured was 20 mU/ml.

EXAMPLE 5

The experiments described in Example 5 were repeated, except that thecells used were LPL-deficient cat fibroblast cells or HepG-2 livercells. Table 1 shows the infection efficiencies at various virusdilutions which were determined for these cell types as well as the CHOK-1 cells.

                  TABLE 1    ______________________________________           DILUTION    VIRUS    2000×                       1000×                               500×                                      250×                                           125×    ______________________________________    CHO K-1  2         5       15     30   50    Cat Fibroblast             10        20      50     100  100    HepG-2   20        50      100    100  100    ______________________________________

Table 2 shows the LPL activity measured for Cat fibroblast cells, andthe LPL mass measured for cat fibroblast cells and HepG-2 cells. Inaddition, Table 2 shows positive control results for COS EV101 cellswhich are over producers of LPL. It can be seen from this data thatthere is a substantial increase in the plasmid activity and also in theamount of the active dimer form of the enzyme.

                  TABLE 2    ______________________________________                  LPL                  Activity                         LPL MASS (ng/ml)    Cell Type             plasmid    (mU/ml)  total                                      monomer dimer    ______________________________________    CHO K-1  control    12       n.d. n.d.    n.d.             pRc/CMV-   20       n.d. n.d.    n.d.             LPL    Cat Fibroblasts             control    0.15      26  24      2             pRc/CMV-   1.5      128  88      34             LPL    HepG-2   control    n.d.      33  28      6             pRc/CMV-   n.d.     164  113     51.5             LPL    COS      EV101      50       530  87      443    ______________________________________

EXAMPLE 6

Vectors for introducing human LDL cDNA into mammalian cells were madeusing the murine leukemia retroviral backbones M3neo, M5neo and JZen1which contain long terminal repeat (LTR) regulatory sequences for themyeloproliferative sarcoma virus. To generate the vectors M3neoLPL andM5neoLPL, a 1.56 kb DraI-EcoRI fragment encompassing the entire LPLamino acid coding region was subcloned into a unique BamHI site located3' or 5' to the neomycin phosphotransferase (neo^(f)), respectively.Expression of both genes is LTR driven in these vectors; in M3neoLPL,functional LPL message would derive from the spliced proviraltranscripts whereas for M5neoLPL, LPL message would derive from the fulllength unspliced proviral transcript. To construct JZenLPLtkneo, a 1092bp Xho I/SalI fragment for neo^(r) was isolated from pMCIneo andinserted into the SalI site of the plasmid pTZ19R, containing the herpessimplex virus thymidine kinase (tk) promoter. The SmaI/HindIII tkneofragment from the pTZ19R was inserted into the Hpa I/Hind III site ofJZen1. A 1.56 kb human LPL cDNA sub-fragment was then cloned in theBamHI site of JZentkneo. Human LPL cDNA was also subcloned directly intoJZen1 to construct JZenLPL.

Virus producer cells lines were then made for each of the viralconstructs using the amphotropic retroviral packaging cell line GP-Am12and the ecotropic packaging line GP-E86. Both cell lines were culturedin HXM medium, which is Dulbecco's modified Eagle's medium (DME)supplemented with 10% heat-inactivated (55° C. for 20 minutes) newborncalf serum (Gibco-BRL), hypoxanthine (15 μg/ml), xanthine (250 μg/ml)and mycophenolic acid (25 μg/ml). For GP-AM12 cells, hygromycin B (200μg/ml) was also added to the HXM medium. All cells were cultured at 37°C. in a humidified atmosphere of 5% CO₂.

EXAMPLE 7

A variety of hematopoietic cell lines were tested using the neomycinresistance marker incorporated in the vector to determine whethertransduction occurred as a result of coincubation with M3neoLPL invitro. K562 erythroid cells, HL60 myeloid cells, and U937 and THP-1monocytic cells obtained from the American Type Culture Collection weregrown in RPMI 1640 medium containing 10% fetal bovine serum. The cellswere then infected by cocultivation (24-48 hours) with irradiated (15 Gyx-ray) near confluent producer cells with polybrene 4 μg/ml added to theco-cultivation medium (RPMI/10% fetal bovine serum). After the infectionperiod, the hematopoietic target cells were maintained in suspensionculture for 24 hours before selection in 1 mg/ml G418. The gene transferefficiencies observed are summarized in Table 3.

The mass of LPL produced was determined for each of the transducedhematopoietic cells lines using two ELISAs. The antibodies used were MAb5D2 which binds to the bioactive dimeric form of LPL and MAb 5F9 whichbinds to both the bioactive dimer and the inactive monomeric form ofLPL. The results are summarized in Table 3.

Finally media supernatants were measured for LPL bioactivity. Theresults of this study are also reported in Table 3.

                  TABLE 3    ______________________________________             Gene Transfer Increase in                                     Increase in    Cell Line             Efficiency    Bioactivity                                     LPL Dimer    ______________________________________    K562     57%           11-fold    5-fold    HL60     47%            9-fold    3-fold    U937     45%           14-fold   54-fold    THP-1    41%            4-fold    2-fold    ______________________________________

These results demonstrate that for each cell type, good transductionefficiencies were achieved, and production of functional LPL resulted.

Transduced HL60 and THP-01 cells were differentiated in macrophages byexposing the cells to 10ng/ml of phorbal ester, PdBU (Phorbol12,13-dibutyrate) for 5 days. For HL60 cells, the LPL bioactivityincreased a further 1.8-fold, while the amount of LPL dimer increasedanother 1.8-fold. No further increase was observed upon differentiationof THP-1 cells.

EXAMPLE 8

NIH 3T3 murine fibroblasts were grown in DME medium containing 10%(vol/vol) fetal bovine serum. The medium on near confluent 60 mm tissueculture plates of viral producer cells 24 hours prior to the plannedinfection with 10 ml DME/10% newborn calf serum. This medium was removedat the time of infection, concentrated 10-fold to a 1.0 ml final volumeby filter centrifugation in Centriprep-30 tubes (Amicon) and diluted 1:4with DME/10% fetal bovine serum with 4 μg/ml polybrene added.Fibroblasts were added to this preparation and incubated for 24-48 hoursat 37° C. 24 hours after viral exposure, cells were subjected toselection in 1.0 mg/ml G418 and grown to confluence. Testing for LPLproduction revealed a 16-fold increase in total LPL production aboveconstitutive levels which consisted almost entirely of dimeric protein,and a 10-fold increase in secreted LPL bioactivity.

EXAMPLE 9

The experiment of Example 8 was repeated using primary human fibroblastcells, FC 1898 and FC 1901 from diagnostic skin biopsies. No measurablelevels of endogenous LPL protein mass or bioactivity could be detectedprior to retroviral-mediated LPL gene delivery. Post transduction levelsof total LPL mass were massively elevated at least 400 times abovenormal. However, at least 82% of this exogenous LPL protein was of theinactive monomeric form. At least a 52-fold (74.8±22/9) increase indimeric LPL production was seen with significantly elevated secretion ofbioactive LPL, approximately 24 times higher (26.9±3.0) than backgroundLPL levels.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: no    (iv) ANTI-SENSE: no    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human    (ix) FEATURE: Primer for exon 6 of human LPL    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GCCGAGATACAATCTTGGTG20    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: no    (iv) ANTI-SENSE: yes    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human    (ix) FEATURE: Mismatch primer for exon 6 of human LPL    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CTGCTTCTTTTGGCTCTGACTGTA24    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: no    (iv) ANTI-SENSE: no    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human    (ix) FEATURE: internal fragment from normal human LPL gene spanning    amino acid 291    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GAGATCAATAAAGTC15    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: no    (iv) ANTI-SENSE: no    (vi) ORIGINAL SOURCE:    (A) ORGANISM: human    (ix) FEATURE: internal fragment from Asn291Ser mutant human LPL gene    spanning amino acid 291    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GAGATCAGTAAAGTC15    __________________________________________________________________________

We claim:
 1. A method for evaluating susceptibility of a humanindividual to premature atherosclerosis comprising the steps of:(a)obtaining a sample of DNA from the individual; and (b) evaluating thesample of DNA for the presence of nucleotides encoding a serine residueat amino acid 291 of the lipoprotein lipase gene product, wherein thepresence of a serine residue is indicative of increased susceptibilityto premature atherosclerosis.
 2. A method according to claim 1, whereinthe sample is amplified using a mismatched primer to form an amplifiedproduct, and the evaluation of step (b) is performed on the amplifiedproduct which is evaluated for a restriction fragment lengthpolymorphism.
 3. A method according to claim 2, wherein the mismatchedprimer binds to the coding strand of the human lipoprotein lipase geneand wherein the base complementary to base number 1130 in the codingstrand is changed from the normal thymine to guanine.
 4. A methodaccording to claim 2, wherein the mismatched primer binds to thenon-coding strand of the human lipoprotein lipase gene and wherein thebase complementary to base number 1130 in the non-coding strand ischanged from the normal adenine to cytosine.
 5. A method according toclaim 2, wherein the mismatched primer has the sequence

    CTGCTTCTTT TGGCTCTGAC TGTA                                 (SEQ 2).


6. A method according to claim 1, wherein the sample is amplified toincrease the amount of genetic material encoding for amino acid 291 ofhuman lipoprotein lipase prior to evaluating the sample for the presenceof nucleotides encoding a serine residue at amino acid
 291. 7. A methodaccording to claim 6, wherein the sample is amplified by a polymerasechain reaction.
 8. A method according to claim 6, wherein the sample isamplified by strand displacement amplification.
 9. A method according toclaim 6, wherein the amplified sample is evaluated for the presence ofnucleotides encoding a serine residue at amino acid 291 by combining thesample with a sequence specific oligonucleotide probe which iscomplementary to a region including the nucleotides encoding amino acid291 of the lipoprotein lipase gene product when amino acid 291 isserine, and wherein the formation of a DNA duplex between the probe andthe amplified sample is determined.
 10. A method according to claim 6,wherein the amplified sample is evaluated for the presence ofnucleotides encoding a serine residue at amino acid 291 by combining thesample with a sequence specific oligonucleotide probe which iscomplementary to a region including the nucleotides encoding amino acid291 of the lipoprotein lipase gene product when amino acid 291 isasparagine, and wherein the formation of a DNA duplex between the probeand the amplified sample is determined.
 11. A method according to claim1, wherein the sample is evaluated for the presence of nucleotidesencoding a serine residue at amino acid 291 by combining the sample witha sequence specific oligonucleotide probe which is complementary to aregion including the nucleotides encoding amino acid 291 of thelipoprotein lipase gene product when amino acid 291 is serine, andwherein the formation of a DNA duplex between the probe and geneticmaterial in the sample is determined.
 12. A method according to claim 1,wherein the sample is evaluated for the presence of nucleotides encodinga serine residue at amino acid 291 by combining the sample with asequence specific oligonucleotide probe which is complementary to aregion including the nucleotides encoding amino acid 291 of thelipoprotein lipase gene product when amino acid 291 is asparagine, andwherein the formation of a DNA duplex between the probe and geneticmaterial in the sample is determined.
 13. A method for evaluating thecausation of premature atherosclerosis in a human patient diagnosed ashaving premature atherosclerosis, comprising the steps of:(a) obtaininga sample of DNA from the individual; and (b) evaluating the sample ofDNA for the presence of nucleotides encoding a serine residue at aminoacid 291 of the lipoprotein lipase gene product, wherein the presence ofa serine residue is indicative of increased susceptibility to prematureatherosclerosis.
 14. A method for evaluating a human individual for riskof coronary artery disease, comprising the steps of(a) obtaining asample of DNA from the individual; and (b) evaluating the sample of DNAfor the presence of a serine residue at amino acid 291 of thelipoprotein lipase gene, wherein the presence of a serine residue isindicative of increased risk of coronary artery disease.
 15. A kit forevaluating the susceptibility of a human individual to prematureatherosclerosis, comprising in packaged combination a pair of primersselected to amplify a region of a human lipoprotein lipase gene encodingfor amino acid 291 of human lipoprotein lipase, wherein one primer ofthe pair of primers is a mismatch primer effective in combination withthe bases encoding amino acid 291 to introduce a restriction site intothe amplified lipoprotein lipase gene, said restriction site beingdifferentially present in amplified normal lipoprotein lipase genes andin amplified lipoprotein lipase genes which have a mutation in the basesencoding amino acid
 291. 16. A kit according to claim 15, wherein themismatch primer binds to the coding strand of the human lipoproteinlipase gene and wherein the base complementary to base number 1130 inthe coding strand is changed from the normal thymine to guanine.
 17. Akit according to claim 16, further comprising a container of therestriction endonuclease RsaI.
 18. A kit according to claim 15, whereinthe mismatch primer binds to the non-coding strand of the humanlipoprotein lipase gene and wherein the base complementary to basenumber 1130 of the non-coding strand is changed from the normal adenineto cytosine.
 19. A kit according to claim 18, further comprising acontainer of the restriction endonuclease RsaI.
 20. A kit according toclaim 14, wherein the mismatch primer has the sequence

    CTGCTTCTTT TGGCTCTGAC TGTA                                 (SEQ 2).


21. A kit for evaluating the susceptibility of a human individual topremature atherosclerosis comprising in packaged combination a pair ofprimers selected to amplify a region of a human lipoprotein lipase geneencoding for amino acid 291 of human lipoprotein lipase and anoligonucleotide probe selected to bind specifically to a portion of thegene encoding human lipoprotein lipase gene, wherein said probe binds toa portion of the gene including the bases coding for amino acid
 291. 22.A kit according to claim 21, wherein the oligonucleotide probe isselected to bind specifically to a gene encoding mutant humanlipoprotein lipase gene having a serine as amino acid 291, wherein saidprobe binds to a portion of the gene including the bases coding for theserine at amino acid
 291. 23. A kit according to claim 22, wherein theprimer pair is adapted for use in a strand displacement amplificationtechnique.
 24. A kit according to claim 21, wherein the oligonucleotideprobe is selected to bind specifically to a gene encoding a normal humanlipoprotein lipase gene having an asparagine as amino acid 291, whereinsaid probe binds to a portion of the gene including the bases coding forthe asparagine at amino acid
 291. 25. A kit for evaluating thesusceptibility of a human individual to premature atherosclerosis orrisk, comprising in packaged combination a pair of primers selected toamplify a region of a human lipoprotein lipase gene encoding for aminoacid 291 of human lipoprotein lipase wherein one of the primers isselected to bind to a region of the human lipoprotein lipase gene whichincludes the bases encoding for amino acid
 291. 26. A kit according toclaim 25, wherein the primer which binds to the region of the humanlipoprotein gene which include the bases encoding for amino acid 291 iscomplementary to a sequence which encodes for serine as amino acid 291.27. A kit according to claim 25, wherein the primer which binds to theregion of the human lipoprotein gene which include the bases encodingfor amino acid 291 is complementary to a sequence which encodes forasparagine as amino acid
 291. 28. A kit for evaluating thesusceptibility of a human individual to premature atherosclerosis,comprising in packaged combination a primer adapted for stranddisplacement amplification of a region of a human lipoprotein lipasegene encoding for amino acid 291 of human lipoprotein lipase, and anoligonucleotide probe having a detectable label attached thereto, saidoligonucleotide probe being selected to bind specifically to the regionof a human lipoprotein lipase gene encoding for amino acid 291 of humanlipoprotein lipase.
 29. A kit according to claim 28, wherein theoligonucleotide probe is selected to bind specifically to DNA encodingfor serine as amino acid
 291. 30. A kit according to claim 28, whereinthe oligonucleotide probe is selected to bind specifically to DNAencoding for asparagine as amino acid
 291. 31. A kit for evaluating ahuman individual for risk of coronary artery disease, comprising inpackaged combination a pair of primers selected to amplify a region of ahuman lipoprotein lipase gene encoding for amino acid 291 of humanlipoprotein lipase, wherein one primer of the pair of primers is amismatch primer effective in combination with the bases encoding aminoacid 291 to introduce a restriction site into the amplified lipoproteinlipase gene, said restriction site being differentially present inamplified normal lipoprotein lipase genes and in amplified lipoproteinlipase genes which have a mutation in the bases encoding amino acid 291.32. An oligonucleotide probe selected to bind specifically to a geneencoding a mutant human lipoprotein lipase having a serine residue asamino acid 291, wherein said probe binds to a portion of the geneincluding the bases coding for the serine residue.
 33. Anoligonucleotide probe according to claim 32, further comprising adetectable label.
 34. An oligonucleotide probe selected to bindspecifically to a gene encoding a normal human lipoprotein lipase havingan asparagine residue as amino acid 291, wherein said probe binds to aportion of the gene including the bases coding for the asparagineresidue.
 35. An oligonucleotide probe according to claim 34, furthercomprising a detectable label.
 36. An oligonucleotide primer which bindsspecifically to a gene encoding for human lipoprotein lipase in a regionadjacent to, but not overlapping the second base in the codoncorresponding the residue 291 in human lipoprotein lipase, said primerincluding a mismatched base which does not correspond to the normalsequence of human lipoprotein lipase, whereby upon extension of theprimer using a target human lipoprotein lipase gene as a template anextension product is produced which contains a restriction site whichcan be cleaved by a restriction endonuclease when the lipoprotein lipaseproduct made by the target gene has a serine residue as amino acid 291,and does not contain such a restriction site when the lipoprotein lipaseproduct made by the target gene has an asparagine residue as amino acid291.
 37. A primer according to claim 36, wherein the primer binds to thecoding strand of the human lipoprotein lipase gene and wherein the basecomplementary to base number 1130 of the coding strand is changed fromthe normal thymine to guanine.
 38. A primer according to claim 36,wherein the primer binds to the non-coding strand of the humanlipoprotein lipase gene and wherein the base complementary to basenumber 1130 of the non-coding strand is changed from the normal adenineto cytosine.
 39. A primer according to claim 36, wherein the primer hasthe sequence

    CTGCTTCTTT TGGCTCTGAC TGTA                                 (SEQ 2).